Therapeutic effects of bryostatins, bryologs, and other related substances on ischemia/stroke-induced memory impairment and brain injury

ABSTRACT

The invention provides for the use of protein kinase activators or boosters of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) or other neurotrophic factors to treat stroke. Specifically, the present invention provides methods of treating stroke comprising the steps of identifying a subject having suffered a stroke and administering to said subject an amount of a pharmaceutical composition comprising a protein kinase C (PKC) activator or 4-methylcatechol acetic acid (MCBA) and a pharmaceutically acceptable carrier effective to treat at least one symptom of stroke.

This application claims benefit to U.S. Provisional Application Ser. No.60/900,339, filed on Feb. 9, 2007 and U.S. Provisional Application Ser.No. 60/924,662, filed on May 24, 2007, all of which are herebyincorporated herein by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to the treatment of stroke with compoundsthat activate protein kinase C (PKC) or boost nerve growth factor (NGF),brain-derived neurotrophic factor (BDNF) or other neurotrophic factors.

BACKGROUND OF THE INVENTION A. Stroke

A stroke, also known as cerebrovascular accident (CVA), is an acuteneurological injury in which the blood supply to a part of the brain isinterrupted. Blood supply to the brain may be interrupted in severalways, including occlusion (ischemic, embolic or thrombotic stroke) orblood-vessel rupture (hemorrhagic stroke). A stroke involves the suddenloss of neuronal function due to disturbance in cerebral perfusion. Thisdisturbance in perfusion is commonly arterial, but can be venous.

The part of the brain with disturbed perfusion no longer receivesadequate oxygen. This initiates the ischemic cascade which causes braincells to die or be seriously damaged, impairing local brain function.Stroke is a medical emergency and can cause permanent neurologic damageor even death if not promptly diagnosed and treated. It is the thirdleading cause of death and the leading cause of adult disability in theUnited States and industrialized European nations. On average, a strokeoccurs every 45 seconds and someone dies every 3 minutes. Of every 5deaths from stroke, 2 occur in men and 3 in women.

Despite the medical emergency and the multiple agents that have beenshown to be effective in arresting the pathological processes ofcerebral ischemia in preclinical studies, thromobolytic therapy usingrTPA is currently the only option available for the treatment ofischemic stroke. The treatment is designed to achieve early arterialrecanalization, which is time-dependent (within 3 hours after the eventto be effective). The effectiveness of rTPA and other potential agentsfor arresting infarct development, depends on early administration oreven before the ischemic event, if possible. The narrow therapeutic timewindow in treating ischemic stroke leads to about only 5% of candidatepatients receiving effective intravenous thrombolytic therapy.

Significant brain injury occurs in ischemic stroke after the immediateischemic event. The “delayed” brain injury and cell death in cerebralischemia/stroke is a well-established phenomenon, representing atherapeutic opportunity. Neurons in the infarction core of focal, severestroke are immediately dead and cannot be saved by pharmacologicintervention. The ischemic penumbra, consisting of the brain tissuearound the core in focal ischemic stroke, and the sensitiveneurons/network in global cerebral ischemia, however, are maintained bya diminished blood supply. The damage to this penumbral brain tissueoccurs in a “delayed” manner, starting 4-6 hours as the second phase ordays and weeks later as the the so-called third phase, after cerebralischemia/stroke. After an about 15 minute cerebral ischemia, forexample, the hippocampal CA1 pyramidal cells start to degenerate within2-3 days, and reach the maximal extent of cell death a week after theischemic event. The sensitive neuronal structures in global cerebralischemia and the ischemic penumbra are “at-risk” tissues. Their salvagethrough intervention or further damage in the subsequent days or weeksdetermine dramatic differences in long-term disability.

The present invention provides a new therapeutic strategy comprising thetransient, periodic or chronic administration of a PKC activator, othercompounds and combinations thereof, to a subject suffering from cerebralischemia/stroke over a broader therapeutic window such as from withinhours to days to weeks, after the ischemic event.

B. Protein Kinase C

PKC has been identified as one of the largest gene families ofnon-receptor serine-threonine protein kinases. Since the discovery ofPKC in the early eighties by Nishizuka and coworkers (Kikkawa et al.(1982) J. Biol. Chem. 257: 13341), and its identification as a majorreceptor for phorbol esters (Ashendel et al. (1983) Cancer Res., 43:4333), a multitude of physiological signaling mechanisms have beenascribed to this enzyme. The intense interest in PKC stems from itsunique ability to be activated in vitro by calcium and diacylglycerol(and its phorbol ester mimetics), an effector whose formation is coupledto phospholipid turnover by the action of growth and differentiationfactors.

The activation of PKC has been shown to improve learning and memory.(U.S. Patent Application Ser. Nos. PCT/US02/13784; PCT/US03/07102;60/287,721; 60/362,081; Ser. Nos. 10/172,005; and 10/476,459; eachincorporated herein by reference in its entirety). Prior to the presentdisclosure, however, the PKC-mediated improvement of learning and memoryhas not been recognized as a mechanism for the treatment of post-strokememory deficits and brain injury. Also, the PKC activators disclosedherein, specifically those compounds that improve learning and memory,were not recognized as possessing brain function-restoring activityafter cerebral ischemia/stroke.

Stroke therapy has historically been limited to few treatment optionsavailable. The only drug therapy currently available, for instance,consists of antithrombotics (thrombolytic therapy; such as intravenousinjections of tissue plasminogen activator), which have to beadministered within 3 hours of the ischemic event. Although many typesof potential neuroprotectants have been tested in clinical trials, nonehas been approved for clinical use, because of ineffectivenessespecially when used post-stroke or associated toxicity. The compoundspresented in this invention disclosure were effective when the treatmentwas started 24 hours after the ischemia in the animal model at dosesthat have already been demonstrated to be well tolerated in humans (thebryostatin-1 doses).Compounds that target the protein kinase C (PKC)such as bryostatin-1, a direct PKC activator, and methylcatecholdiacetic acid, a derivative of methylcatechol, an enhancer or means ofactivating or mobilizing nerve growth factor (NGF), brain-derivedneurotrophic factor (BDNF) or other neurotrophic factors, which isperhaps one of the PKC targets, have been found to have therapeuticvalue against brain injury and memory impairment induced with cerebralischemia in rats (an animal stroke model). T he development of thesesubstances as therapeutic in the treatment of stroke is provided by thisinvention.

SUMMARY OF THE INVENTION

The present invention provides methods of treating stroke comprising thesteps of identifying a subject having suffered a stroke andadministering to said subject an amount of a pharmaceutical compositioncomprising a protein kinase C (PKC) activator or 4-methylcatechol aceticacid (MCBA) and a pharmaceutically acceptable carrier effective to treatat least one symptom of stroke.

In one embodiment, the PKC activator is FGF-18, a macrocyclic lactone, abenzolactam, a pyrrolidinone, or a combination thereof. In a preferredembodiment, the macrocyclic lactone is a bryostatin or neristatin. Inanother embodiment, the neristatin is neristatin-1. In anotherembodiment, the bryostatin is bryostatin-1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17 or 18. More preferably, the bryostatin isbryostatin-1.

In another preferred embodiment, the pharmaceutical compositioncomprises 4-methylcatechol acetic acid (MCBA), other derivatives ofmethylcatechol, or a brain derived neurotrophic factor. MCBA and otherderivatives of methylcatechol activate or upregulate nerve growth factor(NGF), brain derived neurotrophic factor (BDNF) or other neurotrophicfactors. NGF activates, upregulates or enhances the activity of PKCwhich in turn upregulates, activates or enhances NGF.

In one embodiment, administration of the pharmaceutical compositions ofthe present invention is initiated within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, or 14 days of said stroke. In another embodiment, saidadministration is initiated between 1 and 2 days, 1 and 3 days, 1 and 4days, 1 and 5 or 1 and 7 days of said stroke. In another embodiment, theadministration of the pharmaceutical compositions of the presentinvention is initiated within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours of said stroke. Inyet another embodiment, the administration of the pharmaceuticalcompositions of the present invention is initiated between 1 and 3, 1and 5, 1 and 10, 1 and 24, 3 and 5, 3 and 10, 3 and 24, 5 and 10, 5 and24, or 10 and 24 hours after said stroke. In yet another embodiment, theadministration of the pharmaceutical compositions of the presentinvention is initiated after 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours after saidstroke/ischemic event. In yet another embodiment, the administration ofthe pharmaceutical compositions of the present invention is initiatedafter 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, or 21 days after said stroke/ischemic event.

In one embodiment, treatment comprising the administration of thepharmaceutical compositions of the present invention is continued for aduration of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1 depicts a spatial water maze performance of rats over trainingtrials. Data are shown as means±SEM. Bry, bryostatin-1; Isch, cerebralischemia; MCDA, 4-methylcatechol-diacetic acid.

FIG. 2 depicts target quadrant ratio during probe test. Bry,bryostatin-1; Isch, ischemia; MCDA, 4-methylcatechol-diacetic acid *:p<0.05. NS: p>0.05.

DETAILED DESCRIPTION OF THE INVENTION A. Definitions

As used herein, “administration” of a composition includes any route ofadministration, including oral subcutaneous, intraperitoneal, andintramuscular.

As used herein, “an effective amount” is an amount sufficient to reduceone or more symptoms associated with a stroke.

As used herein, “protein kinase C activator” or “PKC activator” means asubstance that increases the rate of the reaction catalyzed by proteinkinase C by binding to the protein kinase C.

As used herein, the term “subject” means a mammal.

As used herein, the term “pharmaceutically acceptable carrier” means achemical composition with which the active ingredient may be combinedand which, following the combination, can be used to administer theactive ingredient to a subject. As used herein, the term“physiologically acceptable” ester or salt means an ester or salt formof the active ingredient which is compatible with any other ingredientsof the pharmaceutical composition, which is not deleterious to thesubject to which the composition is to be administered.

As used herein, “pharmaceutically acceptable carrier” also includes, butis not limited to, one or more of the following: excipients; surfaceactive agents; dispersing agents; inert diluents; granulating anddisintegrating agents; binding agents; lubricating agents; sweeteningagents; flavoring agents; coloring agents; preservatives;physiologically degradable compositions such as gelatin; aqueousvehicles and solvents; oily vehicles and solvents; suspending agents;dispersing or wetting agents; emulsifying agents, demulcents; buffers;salts; thickening agents; fillers; emulsifying agents; antioxidants;antibiotics; antifungal agents; stabilizing agents; and pharmaceuticallyacceptable polymeric or hydrophobic materials. Other “additionalingredients” which may be included in the pharmaceutical compositions ofthe invention are known in the art and described, for example in Genaro,ed., 1985, Remington's Pharmaceutical Sciences, Mack Publishing Co.,Easton, Pa., which is incorporated herein by reference.

The formulations of the pharmaceutical compositions described herein maybe prepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with a carrier or one ormore other accessory ingredients, and then, if necessary or desirable,shaping or packaging the product into a desired single- or multi-doseunit.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for ethical administration to humans, it will be understood bythe skilled artisan that such compositions are generally suitable foradministration to animals of all sorts. Modification of pharmaceuticalcompositions suitable for administration to humans in order to renderthe compositions suitable for administration to various animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and perform such modification with merely ordinary, if any,experimentation. Subjects to which administration of the pharmaceuticalcompositions of the invention is contemplated include, but are notlimited to, humans and other primates, and other mammals.

Despite progress toward the development of new therapeutic agents andavailability of several animal models, there is still a pressing needfor improved animal models for screening

B. Protein Kinase C (PKC)

The PKC gene family consists presently of 11 genes which are dividedinto four subgroups: I) classical PKCα, β₁, β₂ (β₁ and β₂ arealternatively spliced forms of the same gene) and γ, 2) novel PKCδ, ε,η, and θ, 3) atypical PKCζ, λ, η and i and 4) PKC μ. PKC μ resembles thenovel PKC isoforms but differs by having a putative transmembrane domain(reviewed by Blohe et al. (1994) Cancer Metast. Rev. 13: 411; Ilug etal. (1993) Biochem J. 291: 329; Kikkawa et al. (1989) Ann. Rev. Biochem.58: 31). The α, β₁, β₂ and γ isoforms are C²⁺, phospholipid anddiacylglycerol-dependent and represent the classical isoforms of PKC,whereas the other isoforms are activated by phospholipid anddiacylglycerol but are not dependent on Ca²⁺. All isoforms encompass 5variable (V1-V5) regions, and the α, β and γ isoforms contain four(C1-C4) structural domains which are highly conserved. All isoformsexcept PKC α, β and γ lack the C2 domain, the λ η and isoforms also lacknine of two cysteine-rich zinc finger domains in CI to whichdiacylglycerol binds. The Cl domain also contains the pseudosubstratesequence which is highly conserved among all isoforms, and which servesan autoregulartory function by blocking the substrate-binding site toproduce an inactive conformation of the enzyme (House et al. (1987)Science 238, 1726).

100271 Because of these structural features, diverse PKC isoforms arethought to have highly specialized roles in signal transduction inresponse to physiological stimuli (Nishizuka (1989) Cancer 10: 1892), aswell as in neoplastic transformation and differentiation (Glazer (1994)Protein Kinase C, J. F. Kuo, ed., Oxford U. Press at pages 171-198). Fora discussion of known PKC modulators see PCT/US97/08141, U.S. Pat. Nos.5,652,232; 6,080,784; 5,891,906; 5,962,498; 5,955,501; 5,891,870 and5,962,504 (each incorporated herein by reference in its entirety).

There is increasing evidence that the individual PKC isozymes playsignificant roles in biological processes which provide the basis forpharmacological exploitation. One is the design of specific (preferably,isozyme specific) activators of PKC. This approach is complicated by thefact that the catalytic domain is not the domain primarily responsiblefor the isozyme specificity of PKC. These may provide a way to overridethe effect of other signal transduction pathways with oppositebiological effects. Alternatively, by inducing down-regulation of PKCafter acute activation, PKC activators may cause long term antagonism.Bryostatin is currently in clinical trials as an anti-cancer agent. Thebryostatins are known to bind to the regulatory domain of PKC and toactivate the enzyme. Bryostatins are examples of isozyme-selectiveactivators of PKC. (see for example WO 97/43268; incorporated herein byreference in its entirety). For a discussion of known PKC modulators seePCT/US97/08141, U.S. Pat. Nos. 5,652,232; 6,043,270; 6,080,784;5,891,906; 5,962,498; 5,955,501; 5,891,870 and 5,962,504 (each of whichis incorporated herein by reference in its entirety).

Several classes of PKC activators have been identified. Phorbol esters,however, are not suitable compounds for eventual drug developmentbecause of their tumor promotion activity, (Ibarreta et al. (1999) NeuroReport 10(5&6): 1035-40). Of particular interest are macrocycliclactones (i.e. bryostatin class and neristatin class) that act tostimulate PKC. Of the bryostatin class compounds., bryostatin-1 has beenshown to activate PKC and proven to be devoid of tumor promotionactivity. Bryostatin-1, as a PKC activator, is also particularly usefulsince the dose response curve of bryostatin-1 is biphasic. Additionally,bryostatin-1 demonstrates differential regulation of PKC isozymes,including PKCα, PKCδ and PKCε. Bryostatin-1 has undergone toxicity andsafety studies in animals and humans and is actively investigated as ananti-cancer agent. Bryostatin-1's use in the studies has determined thatthe main adverse reaction in humans is myalgia. One example of aneffective dose is 40 μg/m² per week by intravenous injection.

Macrocyclic lactones, and particularly bryostatin-1 is described in U.S.Pat. No. 4,560,774 (incorporated herein by reference in its entirety).Macrocyclic lactones and their derivatives are described elsewhere inU.S. Pat. No. 6,187,568, U.S. Pat. No. 6,043,270, U.S. Pat. No.5,393,897, U.S. Pat. No. 5,072,004, U.S. Pat. No. 5,196,447, U.S. Pat.No. 4,833,257, and U.S. Pat. No. 4,611,066 (incorporated herein byreference in its entirety). The above patents describe various compoundsand various uses for macrocyclic lactones including their use as ananti-inflammatory or anti-tumor agent. (Szallasi et al. (1994) Journalof Biological Chemistry 269(3): 2118-24; Zhang et al. (1996) CanerResearch 56: 802-808; Hennings et al. (1987) Carcinogenesis 8(9):1343-1346; Varterasian et al. (2000) Clinical Cancer Research 6:825-828; Mutter et at (2000) Bioorganic & Medicinal Chemistry 8:1841-1860)(each incorporated herein by reference in its entirety).

As will also be appreciated by one of ordinary skill in the art,macrocyclic lactone compounds and their derivatives, particularly thebryostatin class, are amenable to combinatorial synthetic techniques andthus libraries of the compounds can be generated to optimizepharmacological parameters, including, but not limited to efficacy andsafety of the compositions. Additionally, these libraries can be assayedto determine those members that preferably modulate α-secretase and/orPKC.

Combinatorial libraries high throughput screening of natural productsand fermentation broths has resulted in the discovery of several newdrugs. At present, generation and screening of chemical diversity isbeing utilized extensively as a major technique for the discovery oflead compounds, and this is certainly a major fundamental advance in thearea of drug discovery. Additionally, even after a “lead” compound hasbeen identified, combinatorial techniques provide for a valuable toolfor the optimization of desired biological activity. As will beappreciated, the subject reaction readily lend themselves to thecreation of combinatorial libraries of compounds for the screening ofpharmaceutical, or other biological or medically-related activity ormaterial-related qualities. A combinatorial library for the purposes ofthe present invention is a mixture of chemically related compounds,which may be screened together for a desired property; said librariesmay be in solution or covalently linked to a solid support. Thepreparation of many related compounds in a single reaction greatlyreduces and simplifies the number of screening processes that need to becarried out. Screening for the appropriate biological property may bedone by conventional methods. Thus, the present invention also providesmethods for determining the ability of one or more inventive compoundsto bind to effectively modulate α-secretase and/or PKC.

A variety of techniques are available in the art for generatingcombinatorial libraries described below, but it will be understood thatthe present invention is not intended to be limited by the foregoingexamples and descriptions. (See, for example, Blondelle et al. (1995)Trends Anal. Chem. 14: 83; U.S. Pat. Nos. 5,359,115; 5,362,899; U.S.Pat. No. 5,288,514: PCT publication WO 94/08051; Chen et al. (1994)JACCS 1 6:266 1: Kerr et al. (1993) JACCS 115:252; PCT publicationsW092/10092, W093/09668; W091/07087; and W093/20242; each of which isincorporated herein by reference). Accordingly, a variety of librarieson the order of about 16 to 1,000,000 or more diversomers can besynthesized and screened for a particular activity or property.

Analogs of bryostatin, commonly referred to as bryologs, are oneparticular class of PKC activators that are suitable for use in themethods of the present invention. The following Table summarizesstructural characteristics of several bryologs, demonstrating thatbryologs vary greatly in their affinity for PKC (from 0.25 nM to 10 μM).Structurally, they are all similar. While bryostatin-1 has two pyranrings and one 6-membered cyclic acetal, in most bryologs one of thepyrans of bryostatin-1 is replaced with a second 6-membered acetal ring.This modification reduces the stability of bryologs, relative tobryostatin-1, for example, in both strong acid or base, but has littlesignificance at physiological pH. Bryologs also have a lower molecularweight (ranging from about 600 to 755), as compared to bryostatin-1(988), a property which facilitates transport across the blood-brainbarrier.

PKC Affin Name (nM) MW Description Bryo- 1.35 988 2 pyran + 1 cyclicacetal + macrocycle statin 1 Analog 1 0.25 737 1 pyran + 2 cyclicacetal + macrocycle Analog 2 6.50 723 1 pyran + 2 cyclic acetal +macrocycle Analog 7a — 642 1 pyran + 2 cyclic acetals + macrocycleAnalog 7b 297 711 1 pyran + 2 cyclic acetals + macrocycle Analog 7c 3.4726 1 pyran + 2 cyclic acetals + macrocycle Analog 7d 10000 745 1pyran + 2 cyclic acetals + macrocycle, acetylated Analog 8 8.3 754 2cyclic acetals + macrocycle Analog 9 10000 599 2 cyclic acetals

Analog 1 (Wender et al. (2004) Curr Drug Discov Technol. 1: 1; Wender etal. (1998) Proc Natl Acad Sci USA 95: 6624; Wender et al. (2002) Am ChemSoc. 124: 13648 (each incorporated herein by reference in theirentireties)) possesses the highest affinity for PKC. This bryolog isabout100 times more potent than bryostatin-1. Only Analog 1 exhibits ahigher affinity for PKC than bryostatin. Analog 2, which lacks the Aring of bryostatin-1 is the simplest analog that maintains high affinityfor PKC. In addition to the active bryologs, Analog 7d, which isacetylated at position 26, hasvirtually no affinity for PKC.

B-ring bryologs are also suitable for use in the methods of the presentinvention. These synthetic bryologs have affinities in the low nanomolarrange (Wender et al. (2006) Org Lett. 8: 5299 (incorporated herein byreference in its entirety)). The B-ring bryologs have the advantage ofbeing completely synthetic, and do not require purification from anatural source.

A third class of suitable bryostatin analogs is the A-ring bryologs.These bryologs have slightly lower affinity for PKC than bryostatin 1(6.5, 2.3, and 1.9 nM for bryologs 3, 4, and 5, respectively) but have alower molecular weight.

A number of derivatives of diacylglycerol (DAG) bind to and activateprotein kinase C (Niedel et al. (1983) Proc. Natl. Acad. Sci. USA 80:36; Mori et al. (1982) J. Biochem (Tokyo) 91: 427; Kaibuchi et al.(1983) J. Biol. Chem. 258: 6701). However, DAG and DAG derivatives areof limited value as drugs. Activation of PKC by diacylglycerols istransient, because they are rapidly metabolized by diacylglycerol kinaseand lipase (Bishop et al. (1986) J. Biol. Chem. 261: 6993; Chung et al.(1993) Am. J. Physiol. 265: C927; incorporated herein by reference intheir entireties). The fatty acid substitution determines the strengthof activation. Diacylglycerols having an unsaturated fatty acid are mostactive. The stereoisomeric configuration is also critical. Fatty acidswith a 1,2-sn configuration are active, while 2,3-sn-diacylglycerols and1,3-diacylglycerols do not bind to PKC. Cis-unsaturated fatty acids aresynergistic with diacylglycerols. In one embodiment of the presentinvention, the term “PKC activator” expressly excludes DAG or DAGderivatives, such as phorbol esters.

Isoprenoids are PKC activators suitable for use in the methods of thepresent invention. Farnesyl thiotriazole, for example, is a syntheticisoprenoid that activates PKC with a Kd of 2.5 μM. Farnesylthiotriazole, for example, is equipotent with dioleoylglycerol (Gilbertet al. (1995) Biochemistry 34: 3916; incorporated herein by reference inits entirety), but does not possess hydrolyzable esters of fatty acids.Farnesyl thiotriazole and related compounds represent a stable,persistent PKC activator. Because of its low MW (305.5) and absence ofcharged groups, farnesyl thiotriazole would readily cross theblood-brain barrier.

Octylindolactam V is a non-phorbol protein kinase C activator related toteleocidin. The advantages of octylindolactam V, specifically the(-)-enantiomer, include greater metabolic stability, high potency(Fujiki et al. (1987) Adv. Cancer Res. 49: 223; Collins et al. (1982)Biochem. Biophys. Res. Commun. 104: 1159; each incorporated herein byreference in its entirety)(EC50=29 nM) and low molecular weight thatfacilitates transport across the blood brain barrier.

Gnidimacrin is a daphnane-type diterpene that displays potent antitumoractivity at concentrations of 0.1-1 nM against murine leukemias andsolid tumors. It acts as a PKC activator at a concentration of ≈3 nM inK562 cells, and regulates cell cycle progression at the G1/S phasethrough the suppression of Cdc25A and subsequent inhibition of cyclindependent kinase 2 (Cdk2) (100% inhibition achieved at 5 ng/ml).Gnidimacrin is a heterocyclic natural product similar to bryostatin, butsomewhat smaller (MW=774.9).

Iripallidal is a bicyclic triterpenoid isolated from Iris pallida.Iripallidal displays anti-proliferative activity in a NCI 60 cell linescreen with G150 (concentration required to inhibit growth by 50%)values from micromolar to nanomolar range. It binds to PKCα with highaffinity (Ki=75.6 nM). It induces phosphorylation of ERK1/2 in aRasGRP3-dependent manner. M.W. 486.7. Iripallidal is only about half thesize of bryostatin and lacks charged groups.

Ingenol is a diterpenoid related to phorbol but possesses much lesstoxicity. It is derived from the milkweed plant Euphorbia peplus.Ingenol 3,20-dibenzoate, for example, competes with [3H]phorboldibutyrate for binding to PKC (Ki for binding=240 nM) (Winkler et al.(1995) J. Org. Chem. 60: 1381; incorporated herein by reference).Ingenol-3-angelate possesses antitumor activity against squamous cellcarcinoma and melanoma when used topically (Ogbourne et al. (2007)Anticancer Drugs. 18: 357; incorporated herein by reference).

Napthalenesulfonamides, includingN-(n-heptyl)-5-chloro-1-naphthalenesulfonamide (SC-10) andN-(6-Phenylhexyl)-5-chloro-1-naphthalenesulfonamide, are members ofanother class of PKC activators. SC-10 activates PKC in acalcium-dependent manner, using a mechanism similar to that ofphosphatidylserine (Ito et al. (1986) Biochemistry 25: 4179;incorporated herein by reference). Naphthalenesulfonamides act by adifferent mechanism from bryostatin and would be expected to show asynergistic effect with bryostatin or a member of another class of PKCactivators. Structurally, naphthalenesulfonamides are similar to thecalmodulin (CaM) antagonist W-7, but are reported to have no effect onCaM kinase.

The linoleic acid derivative DCP-LA(2-[(2-pentylcyclopropyl)methyl]cyclopropaneoctanoic acid) is one of thefew known isoform-specific activators of PKC known. DCP-LA selectivelyactivates PKCε with a maximal effect at 100 nM. (Kanno el al. (2006) J.Lipid Res. 47: 1146). Like SC-10, DCP-LA interacts with thephosphatidylserine binding site of PKC, instead of the diacylglycerolbinding site.

An alternative approach to activating PKC directly is to increase thelevels of the endogenous activator, diacylglycerol. Diacylglycerolkinase inhibitors such as6-(2-(4-[(4-fluorophenyl)phenylmethylene]-1-piperidinyl)ethyl)-7-methyl-5H-thiazolo[3,2-a]pyrimidin-5-one(R59022) and[3-[2-[4-(bis-(4-fluorophenyl)methylene]piperidin-1-yl)ethyl]-2,3-dihydro-2-thioxo-4(1H)-quinazolinone(R59949) enhance the levels of the endogenous ligand diacylglycerol,thereby producing activation of PKC (Meinhardt et al. (2002) Anti-CancerDrugs 13: 725).

A variety of growth factors, such as fibroblast growth factor 18(FGF-18) and insulin growth factor, function through the PKC pathway.FGF-18 expression is upregulated in learning and receptors for insulingrowth factor have been implicated in learning. Activation of the PKCsignaling pathway by these or other growth factors offers an additionalpotential means of activating protein kinase C.

Growth factor activators, such as the 4-methyl catechol derivatives,such as 4-methylcatcchol acetic acid (MCBA), that stimulate thesynthesis and/or activation of growth factors such as NGF and BDNF, alsoactivate PKC as well as convergent pathways responsible forsynaptogenesis and/or neuritic branching.

The present compounds can be administered by a variety of routes and ina variety of dosage forms including those for oral, rectal, parenteral(such as subcutaneous, intramuscular and intravenous), epidural,intrathecal, intra-articular, topical and buccal administration. Thedose range for adult human beings will depend on a number of factorsincluding the age, weight and condition of the patient and theadministration route.

All books, articles, patents or other publications and references arehereby incorporated by reference in their entireties. Reference to anycompound herein includes the racemate as well as the single enantiomers.

EXAMPLES

The following Examples serve to further illustrate the present inventionand are not to be construed as limiting its scope in any way.

Example 1 Global Ischemia Model of Stroke

Rats (male, Wistar, 200-225 g) were randomly divided into 6 groups (8each) and housed for 1 week before experimentation. Transient orpermanent restriction of cerebral blood flow and oxygen supply resultsin ischemic stroke. The global ischemia model used to induce vascularmemory impairment was two-vessel occlusion combined with a short termsystemic hypoxia. Ligation of the bilateral common carotid arteries wasperformed under anesthesia (pentobarbital, 60 mg/kg, i.p.). After aone-week recovery from the surgery, rats were exposed to 14-min hypoxia(5% oxygen in a glass jar). Control rats (sham operated and vehiclecontrols) were subjected to the same incision to isolate both commoncarotid arteries and to 14-min air (in the glass jar). Body temperaturewas kept at 37-37.5° C. using a heating light source during the surgicalprocedure and until the animals were fully recovered.

Example 2 Bryostatin and MCDA Treatment

Bryostatin-1 was administered at 20 μg/m² (tail i.v., 2 doses/week, for10 doses), starting 24 hours after the end of the hypoxic event.4-Methylcatechol-diacetic acid (MCDA, a potential NGF and BDNF booster)was administered at 1.0 mg/kg (i.p., daily for the same 5-week period)in separate groups of rats.

One week after the last bryostatin-1, MCDA, or vehicle administration,rats were trained in the water maze spatial learning task (2 trainingtrials per day for 4 days), followed by a probe test. A visible platformtest was given after the probe test. The results are shown in FIG. 1.

Overall, there was a significant learning difference between the 6groups (FIG. 1; F_(5.383)=27.480, p<0.001; ANOVA). Detailed analysisrevealed that the ischemic group did not learn the spatial maze tasksince there was no significant difference in escape latency over trials(F_(7,63)=0.102, p>0.05), a significantly impaired learning as comparedwith the control rats (group difference: F_(1,127)=79.751, p<0.001),while the rats in the other 5 groups all learned the task (the ischemicrats with MCDA treatment: p<0.05 and the other 4 groups: p<0.001 overtrials). Bryostatin-1 therapy greatly improved the performance (Ischemicgroup with bryostatin-1 treatment vs. ischemic rats: F_(1,127)=72.782,p<0.001), to the level of performance that did not differ statisticallyfrom the control rats (Ischemic group with bryostatin-1 treatment vs.control rats: F_(1,127)=0.001, p>0.05). MCDA treatment also improved thelearning of the ischemic rats (ischemia with NCDA treatment vs. ischemicrats: F_(1,127)=15.584, p<0.001) but the difference between the ischemiawith MCDA treatment and control rats remained significant after the 5week treatment (ischemia with NCDA treatment vs. control rats:F_(1,127)=16.618, p<0.001). There were no differences between thecontrol and bryostatin-1-only groups (bryostatin-1 vs. control:F_(1,127)=0.010, p>0.05) and between the control and MCDA-only groups(MCDA vs. control: F_(1,127)=0.272,p>0.05).

The rats in the ischemic group did not show a target preference in theprobe test (F3,31=0.096, p>0.05), while the rats of the other 5 groupsall showed a target quadrant preference in the probe test (all p<0.005).Data were analyzed using target quadrant ratio (dividing the targetquadrant distance by the average of the non-target quadrant valuesduring the probe test; FIG. 2). There was a significant difference inthe target quadrant ratios between the groups (F5,47=5.081, p<0.001).Detailed analysis revealed group differences between the control andischemic rats (F1,15=9.451, p<0.01), between the ischemic and ischemicwith bryostatin-1 treatment (F1,15=10.328, p<0.01), and between theischemic with MCDA treatment and ischemic rats (F1,15=5.623, p<0.05),but no differences between the control and ischemic rats withbryostatin-1 treatment (F1,15=0.013, p>0.05), between the ischemic withMCDA treatment and control groups (F1,15=2.997, p>0.05), between thecontrol and bryostatin-l-only rats (F1,15=0.064, p>0.05), and betweenthe control and the MCDA-only rats (F1,15=0.0392, p>0.05). A visibleplatform test, determined after the probe test revealed no significantdifference between the groups (F5,47=0.115, p>0.05), indicating thatthere were no significant group differences in sensorimotor ability ofthe rats.

Example 3 Bryostatin Treatment

Global cerebral ischemia/hypoxia was induced in male Wistar rats(225-250 g) by permanently occluding the bilateral common carotidarteries, combined with about 14 minutes of low oxygen (about 5%).Bryostatin-1 was administered at 15 μg/m² (via a tail vein, 2doses/week, for 10 doses), starting about 24 hours after the end of theischemic/hypoxic event. Spatial learning (2 trials/ day for 4 days) andmemory (a probe test of 1 minute, 24 hours after the last trial) taskwas performed 9 days after the last dose. Overall, there was asignificant difference between the groups (F3,255=31.856, p<0.001) andgroups x trials (F21,255=1.648, p<0.05). Global cerebral ischemiaimpaired the spatial learning (ischemial vs. sham-operatedF1,127=79.751, p>0.001). The learning impairment was restored byBryostatin-1 treatment (Bryostatin-1+Ischemia vs. Ischemia:F1,127=50.233, p<0.001), while Bryostatin-1 alone did not affect thelearning (Bryostatin-1 vs. sham-operated: F1,127=2.258, p>0.05; 9 daysafter the last dose).

In the memory retention test, sham-operated rats showed a targetquadrant preference. Such good memory retention was not observed in theischemic rats, indicating an impaired spatial memory. Bryostatin-1therapy effectively restored memory retention after ischemia to thelevel of the sham-operated rats. Bryostatin-1 alone had no significanteffects in the target quadrant preference compared with that of thesham-operated control rats. There was a significant difference in thequadrant ratios (calculated by dividing the target quadrant swimdistance by the average swim distance in the non-target quadrants;F3,31=6.181, p<0.005) between the groups. Detailed analysis revealedsignificant differences between the ischemic rats and sham-operatedcontrol rats (F1,15=9.451, p<0.01), between the ischemic rats andischemic rats with Bryostatin-1 treatment (F1,15=10.328, p<0.01), but nosignificant differences between the ischemic rats with Bryostatin-1treatment and sham-operated control (F1,15=0.0131, p>0.05) and betweenthe sham-operated control rats and Bryostatin-1 alone rats (F1,15=0.161,p>0.05). These results demonstrate that the cerebral ischemia/hypoxiaproduced an impairment of spatial learning and memory, tested about 7weeks after the ischemic event. The impairment was lasting and notrecoverable, during the time frame without appropriate intervention, butrestored by chronic Bryostatin-1 treatment, even when the treatment wasstarted 24 hours after the ischemic event, a wide therapeutic timewindow.

1-19. (canceled)
 20. A method of treating in a subject who has sufferedan ischemic event, the method comprising administering to the subject aneffective amount of a pharmaceutical composition sufficient to treat atleast one symptom of stroke, wherein the composition comprises at leastone protein kinase C (PKC) activator and a pharmaceutically acceptablecarrier, and wherein the at least one PKC activator is chosen frombryologs, diacylglycerol derivatives other than phorbol esters,isoprenoids, daphnane-type diterpenes, bicyclic triterpenoids,naphthalenesulfonamides, linoleic acid derivatives, diacylglycerolkinase inhibitors, growth factor activators, and combinations thereof.21. The method of claim 20, wherein the at least one PKC activatorcomprises a growth factor activator chosen from 4-methylcatecholderivatives.
 22. The method of claim 21, wherein the 4-methylcatecholderivative is 4-methylcatechol acetic acid.
 23. The method of claim 20,wherein administration of the pharmaceutical composition is initiatedfrom 1 to 3 days after the ischemic event.
 24. The method of claim 20,wherein the treatment is continued from 1 to 6 weeks.
 25. The method ofclaim 20, wherein the treatment reverses stroke-induced brain injury.26. The method of claim 20, wherein the treatment reversesstroke-induced memory impairment.
 27. The method of claim 20, whereinthe at least one PKC activator comprises a bryolog chosen from a B-ringbryolog and an A-ring bryolog.
 28. The method of claim 27, wherein thebryolog has a molecular weight ranging from about 600 to 755 and anaffinity for PKC ranging from about 0.25 nM to 10 μM.
 29. The method ofclaim 20, wherein the at least one PKC activator comprises the bryolog


30. The method of claim 20, wherein the at least one PKC activatorcomprises the bryolog


31. The method of claim 27, wherein the B-ring byrolog is chosen from

and
 32. The method of claim 27, wherein the A-ring bryolog is chosenfrom

wherein R is t-Bu, Ph, or (CH₂)₃p-Br—Ph.
 33. The method of claim 20,wherein the at least one PKC activator comprises a diacylglycerolderivative comprising unsaturated fatty acids.
 34. The method of claim33, wherein the fatty acids are in a 1,2-sn configuration.
 35. Themethod of claim 33, wherein the fatty acids comprise cis-unsaturatedfatty acids.
 36. The method of claim 20, wherein the at least one PKCactivator comprises octylindolactam V.
 37. The method of claim 36,wherein the octylindolactam comprises the (−)-enantiomer.
 38. The methodof claim 20, wherein the at least one PKC activator comprisesgnidimacrin.
 39. The method of claim 20, wherein the at least one PKCactivator comprises iripallidal.
 40. The method of claim 20, wherein theat least one PKC activator comprises ingenol.
 41. The method of claim20, wherein the at least one PKC activator comprises ingenol3,20-dibenzoate.
 42. The method of claim 20, wherein the at least onePKC activator comprises ingenol-3-angelate.
 43. The method of claim 20,wherein the at least one PKC activator comprises a napthalenesulfonamidechosen from N-(n-heptyl)-5-chloro-1-napthalenesulfonamide andN-(6-Phenylhexyl)-5-chloro-1-naphthalenesulfonamide.
 44. The method ofclaim 20, wherein the at least one PKC activator comprises2-[(2-pentylcyclopropyl)methyl]-cyclopropaneoctanoic acid.
 45. Themethod of claim 20, wherein the at least one PKC activator comprises6-(2-(4-[(4-fluorophenyl)phenylmethylene]-1-piperidinyl)ethyl)-7-methyl-5H-thiazolol[3,2-a]pyrimidin-5-one.46. The method of claim 20, wherein the at least one PKC activatorcomprises[3-[2-[4-(bis-(4-fluorophenyl)methylene]piperidin-1-yl)ethyl]-2,3-dihydro-2-thioxo-4(1H)-quinazolinone.